1. Field of the Invention
This invention relates to compounds used for chemotherapeutic treatment of infections produced by human retroviruses and to methods for their use in treatment of such diseases as acquired immunodeficiency syndrome, AIDS, or AIDS-Related Complex (ARC).
2. Description of Related Art
There have been more than 100,000 deaths from AIDS and ARC to date, yet currently no treatments for the diseases effect cures. The drugs AZT, DDI and DOC have been approved by the Food and Drug Administration, and when used alone or in combination prolong the life of patients, but do not produce cures. Approximately seventy additional compounds are in the early stages of clinical testing, but FDA approval for additional compounds has not been forthcoming.
Many of the presently known compounds are toxic and may be eliminated in humans rapidly, requiring heavy dosage schedules. In addition, most nucleoside analogs, such as AZT, require initial phosphorylation by cellular kinases for activation.
An especially promising target for treatment and prevention of AIDS and ARC is the HIV protease. HIV produces a small, dimeric aspartyl protease which specifically cleaves the polyprotein precursors encoding the structural proteins and enzymes of the virus. This proteolytic activity is absolutely required for the production of mature, infectious virions and is therefore an attractive target for therapeutic intervention. The resolved X-ray crystallographic structures of HIV-1 protease and a handful of HIV-1 protease-inhibitor complexes are available. The active site of this enzyme can be roughly described as an open-ended cylinder which is lined almost exclusively by hydrophobic amino acids (FIG. 1A). Notable exceptions to this hydrophobic trend are the two catalytic aspartic acids (Asp25, Asp125), which catalyze the attack of water on the scissile peptide bond of the substrate. Efforts in molecular genetics, protein biochemistry, enzymology, medical chemistry, virology, X-ray crystallography, molecular modeling have all been marshalled to identify specific inhibitors of this critical viral enzyme (C. Debouck, AIDS Res. and Human Retroviruses, 8:153-164, 1992). Some of these compounds have also demonstrated activity in humans infected with HIV-1, as measured by p24 decline and increased CD4+ cell counts.
The research on buckminsterfullerene (C60) and other fullerenes in the last few years has been extraordinary. Fullerenes are hollow molecules composed of pure carbon atoms. Typically, fullerenes each have 12 pentagons, but differing numbers of hexagons. The pentagons are required in order to allow curvature and eventual closure of the surface upon itself. The most abundant species to date is the C60 molecule known as buckminsterfullerene. Its crystal and molecular structure have been resolved using single-crystal x-ray diffraction methods (S. Liu, et al., Science, 254:408-410, 1991). C60 consists of 12 pentagons and 20 hexagons and is classified as an icosahedron, the highest symmetry structure possible.
Naturally occurring fullerenes have recently been found in the geological environment of Shunga, a town in the lake region of Karwelia in Russia (P. R. Buseck, et al, Science, 247:215-217, 1992). Synthetic fullerenes are produced by high temperature vaporization of solid graphite rods by resistive heating or arc heating in the presence of a few to several torr of rare gas. The soot produced by the vaporization contains varying levels of fullerenes, depending on the vaporization conditions. However, the majority of the fullerenes produced are C60 and C70, with C60 being more abundant. The fullerenes are extracted from the soot by placing the soot into a solvent in which the fullerenes are soluble. The solution is then filtered and allowed to evaporate to yield fullerene powders. Alternatively, the fullerenes can be purchased commercially.
A host of physical and chemical properties of these materials have now been established, and their potential applications in several areas are now apparent. To date, however, no specifically targeted fullerene molecule, with a special biological function in mind, has been prepared. There exist a variety of procedures for functionalization of C60 fullerenes. (See, for example, Fullerenes Synthesis, Properties, and Chemistry of Large Carbon Clusters, G. Hammond, et al., Eds., ACS Symposium Series 481, American Chemical Society, Washington, D.C., 1992; see entire issue No. 3 of Acc. Chem. Res., 25, 1992; A. Hirsch, et al., Chem. Int. Ed. Engl., 31, 766, 1992). Nearly all the fullerenes characterized are nonderivatized homologs (spheroids, tubes, etc.) of C60, and like C60 itself, are highly hydrophobic and insoluble in aqueous media. Recently fullerene-cyclodextrin inclusion compounds comprising C60 embedded in xcex3-cyclodextrin (T. Anderson, et al., J. Chem. Soc. Chem. Commun., 1992:604-606, 1992) and fullerenes containing multiple covalently attached substituents (U.S. Pat. No. 5,177,248) or multiple covalently attached amine-derived substituents (A. Hirsch, et al., Angew. Chem. Int. Ed. Engl., 30:1309-1310, 1991; V. Mehrotra, et al., Chem. Mat., 4:20-22, 1992) have been shown to have water solubility, but the lability of the former, and the configurational dynamism and complex isomerism of the latter compounds would preclude a ready and unequivocal evaluation of structure-activity data in biological systems. Polyhydroxylated, water-soluble fullerenes have also been prepared, but no single, fully characterized isomer has been isolated to date (L. Y. Chiang, et al., J. Chem. Soc. Chem. Commun., 1992:1701-1793, 1992).
The need exists, therefore, for therapeutic compounds useful in ameliorating or preventing retroviral infections, especially AIDS and AIDS-Related Complex (ARC). The present invention provides derivatives of C60 that are water soluble at physiologic conditions and both prophylatically and therapeutically effective against the virus that causes AIDS and ARC.
The ability of water soluble buckminsterfullerene (C60) derivatives to inactivate virus through binding with the active site of HIV-1 protease (HIVP) and/or inhibition of reverse transcriptase has been examined through model building and simple physical chemical analysis. The prediction that these compounds should bind to the active site of the HIVP protease, and thereby act as inhibitors, has been borne out by assay of cells acutely and chronically infected with human immunodeficiency virus (HIV).
The complexes generated via computer models suggest that the virucidal activity of C60 derivatives results from a snug fit of the fullerene into the active site of the HIVP protease, thereby removing at least 298 xc3x852 of primarily nonpolar surface from solvent exposure and driving ligand/protein association. A method for screening the binding of candidate buckministerfullerene derivatives with the active site of HIVP using computer simulation is also provided.
Kinetic analysis of HIVP in the presence of a water-soluble C60 derivative, bis(phenethylamino-succinate), suggests a competitive mode of inhibition. This is consistent with and supports the predicted binding mode.
The water soluble C60 derivatives for inactivating HIV by interacting with HIV by Van der Waals contacts with the nonpolar HIVP surface, are substituted symmetrically with organic moieties comprising from 1 to about 20 carbon atoms each and optionally further comprising polar heteroatoms, such as oxygen and nitrogen. The preferred fullerene derivatives are diamino derivatives synthesized in three steps from C60 via a suitably substituted diphenyldiazomethane. High-resolution mass spectrometric analysis of these compounds is possible only under the mild conditions of matrix-assisted laser desorption/ionization Fourier transform mass spectrometry. Direct infrared or ultraviolet laser desorption degrades the compounds so that only C60 ions are observed, in either positive or negative mode. Virus inactivation assays are provided for confirming activity of the C60 derivatives against human immunodeficiency virus type 1 (HIV-1) and HIV-2. Inhibition of acutely and chronically HIV-1 infected human peripheral blood mononuclear cells (PBMC) and chronically infected lymphocytes (e.g., H9 cells) by the preferred water soluble bis(monosuccinimide) derivative of p,pxe2x80x2-bis(2-aminoethyl)-diphenyl has an EC50 at a concentration of 7 xcexcM, and no cytotoxicity is shown in uninfected PBMC as well as other cell lines. Inhibition of protease (HIVP) and reverse transcriptase (HIVRT) is also shown.